Emilie Rissman - US grants

Hormonal mechanisms that control the expression of reproductive behavior are complex. In the female, estradiol and progesterone are the predominant gonadal steriods which stimulate behavior. Some species, such as the musk shrew, also require adrenal hormone to activate behavior. The mechanisms involved for these behavioral responses are unclear. Moreover, the role of the adrenal hormones in the stimulation of reproductive behavior as opposed to aggressive behavior present a fascinating question. The present proposal contains carefully designed experiments in which the musk shrew's behavioral responses are described and then altered by pharmacological manipulations. Current methodologies will be used to monitor adrenal and gonadal hormones. An elegant technique will be used to sample the animal without disturbing the individual; thereby enabling a "true" measure of the interactions between behavioral and physiological responses. These investigations will significantly advance the field of the hormonal basis of behavior.

The examination of puberty in male mammals, and its regulation by environmental variables has received relatively little experimental attention. A small number of species of temperate zone male rodents have been examined. In these animals nutritional, photoperiodic, and social variables have varying effects on various measures of sexual maturation. However, the vast majority of mammalian species evolved and still live in the tropics. Here, since environmental cues such as temperature and photoperiod are relatively invariant, the most likely regulators of puberty are food availability and social cues. Reproduction is an energetically demanding process, thus food availability and normal growth are critical for sexual maturation. Likewise, mate availability is important. Male maturation can be influenced by the presence, or absence of fertilizable females. Not only are these two variables of interest independent of each other, they also co-vary and undoubtedly modify the effects of each on puberty. I plan to use the male musk shrew (Suncus murinus), a tropical mammal to develop a model system in which to examine the effects of these two,interdependent, environmental cues on male sexual maturation. I will manipulate food and mate availability by restricting males' access to food and/or females. I will also determine the extent to which these two variables are related to influence each other. Unlike past work in this area I plan to examine several behavioral, physiological and hormonal aspects of puberty. This is important since some environmental variables may effect puberty via physiological versus psychological(behavioral) channels. The goal of this proposal is to develop a system in which the effects of both positive and negative environmental cues on male puberty can be assayed independently and dependently.

While it is known that ovarian and adrenal steroid hormones are involved in the regulation of reproductive behaviors, the question remains as to the specific role that androgen and estrogen play. While once considered a straightforward question, it is now complicated by the finding that testosterone, the major androgen, is metabolized into estrogen in peripheral and neural tissues. Since strong evidence is accumulating to suggest a function for androgen in expression of female behavior in both humans and primates, it becomes imperative to know the identity of the hormone mediating each function. Dr. Rissman has developed a model system to examine this important question. She uses members of a small primitive mammalian species of the order of Insectivora which have a number of striking features similar to humans. Dr. Rissman has designed a series of experiments to determine whether androgen is acting directly or by its neural metabolism to estrogen. Once she has this information, Dr. Rissman will define the location in the brain where the hormone is acting. These studies will provide important information towards understanding the underlying neural mechanisms. Moreover, this work could be very beneficial in prescribing hormonal therapy for postmenopausal women and women who have had their ovaries and/or adrenals removed for medical purposes.

The Center for Biological Timing consists of 20 laboratories, 17 of which are at the University of Virginia. This award will renovate laboratory space for five center investigators as well as core facility research and research training space for all investigators. The renovated laboratory space will be located in the Department of Biology and in the multistory building of the Health Science Center. The total amount of renovated space is approximately 12,000 gross square feet and will be used for the latest molecular bio-technology applied to biological timing. The improvement of the laboratory space will greatly enhance the research productivity and research training opportunities in the Center laboratories.

DESCRIPTION: This is a proposal for the continuation of a Research Scientist Development Award, now called an Independent Scientist Award (K02). During the first 4 years of this award the applicant's research program has expanded significantly. The applicant initiated a new line of research involving new collaborations and new technical skills. This program takes advantage of the laboratory mouse as a model system to study behavioral neuroendocrinology. The advantages that the mouse offers are the richness of its behavioral repertoire coupled with a superior knowledge of its genetics, and the ability to design and produce genetically engineered animals. The K02 award allowed the applicant to receive significant new training in neuro- and molecular biological techniques. The applicant's interest in sexual behaviors and the role of aromatization of androgen to estrogen in the activation of these behaviors lead her to begin working with the estrogen receptor alpha knockout mouse (ERaKOs). Her work to date has revealed that although sexual behavior and social preferences are deficient in ERaKO males and females, treatment with the dopamine agonist apomorphine can reinstate normal behavior. This finding challenges the dogma that aromatized estrogens masculinize brain development during the prenatal period. To further evaluate the role of steroid receptors in activation of sexual behavior the PI has obtained several additional KO mice; including the estrogen receptor beta knockout and Tfm mice (androgen receptor knockouts). During the upcoming K02 grant period the PI will examine the molecular bases for sexual behavior using a variety of approaches. These include; 1) in vivo microdialysis to examine neural catecholamine responses to nitric oxide and sexual encounters; 2) the use of high-density DNA arrays to monitor gene expression in response to hormones and/or behavioral interactions; and 3) training in molecular mouse genetics. The K02 award is extremely valuable since it frees the PI from much of her administrative and teaching responsibilities. This allows her to spend significant amounts of time in colleagues' laboratories learning new techniques and focus her energy on running her own program.

behavioral /social science research tag; hormone regulation /control mechanism

DESCRIPTION (provided by applicant): The long term objective of these studies is to understand how genes, hormones, and environment interact and ultimately control complex social behaviors. The human and mouse genomes have been sequenced. Human endocrine mutations are common in the clinic, and engineered and sponateous mouse mutants along with hormone receptors are available for many of the enzymes that regulate hormone sythesis. Thus, the genetic bases of major hormones and their receptors are well known. But the inverse relationship, how behavior affects gene function, is relatively unexplored. The studies proposed here will dissect gene, hormone, and behavior interactions and elucidate the mechanisms by which the steroid hormone receptor, estrogen receptor alpha affects the evolutionarily essential and conserved set of behaviors that consititute male sexual behavior. We will use central administration of dopamine to activate sexual behavior in male mice lacking a functional estrogen receptor alpha gene, and we will ask if sexual experience can compensate for exogenous dopamine. In vivo microdialysis will be conducted to determine if estrogen and/or the gaseous neurotransmitter, nitric oxide, can stimulate dopamine release in the medial preoptic area. In addition, we will ask if the estrogen receptor alpha is required for female-induced dopamine release in the brain and if this is modified by sex experience. To pinpoint which dopamine receptor is essential for this behavior we will use dopamine receptor agonists and antagonists, appropriate doamine receptor knockout mice, and the progestin receptor knockout mouse to ask if dopamine acts via the unoccupied progestin receptor to initiate sexual behavior in naive males. The innovative aspects of this program include the use of pharmacology, knockout mice, and life experiences as factors that affect gene actions. Sexual dysfunctions including premature ejaculation and erectile disorders are common in men. Our understanding of the neurobiology of these disorders is limited, and the interactions between sexual experiential factors and treatment are unknown. Several clinical treatments for erectile disorder in men incorporate the use of drugs we will use for our work; nitric oxide donors and dopamine agonists. The information we will generate may help explain why patients undergoing similar drug or hormone therapies often display wide individual variations in treatment outcomes.

An important compound involved in reproduction in all vertebrate species is gonadotropin-releasing hormone, GnRH. This peptide is produced in nerve cells of the brain, and regulates synthesis and release of pituitary hormones, which in turn stimulate ovulation. The ancestral mammal was a shrew-like animal, and some shrew species today still lack an ovarian or behavioral estrous cycle, and puberty is initiated by mating rather than by ovarian hormones as in most other mammals. In a species called the musk shrew, the first mating triggers rapid changes in GnRH in the forebrain, and multiple matings initiate ovulation. Shrews have very high metabolism, are largely solitary 'opportunistic' breeders, and reproduction is further regulated by local environmental conditions, particularly food availability, independent of sex hormone cycles. In many mammals including primates, there are at least two forms of GnRH, and one may primarily regulate ovulation, and the other may regulate behavior. This project uses immunocytochemical and molecular techniques with the musk shrew as a unique model to explore novel issues of how mating and food intake affect the functions of the two forms of GnRH in the brain, to regulate sexual behavior and puberty.
The impact of this work will extend beyond neuroscience to reproductive endocrinology, and to comparative and evolutionary biology. In addition, this project will support a unique animal colony as a national resource, and will support student training in neuroendocrinology.

Estrogen is a steroid hormone that is important throughout the lifespan. It is involved in functions including cell migration, bone development, reproductive development, neurogenesis, heart function and even neural plasticity and cognition, as well as its well-known actions as a reproductive hormone. Estrogen acts at the cellular level through two main known molecular receptors, alpha (ER-a) and beta (ER-B). These receptors are transcription factors that promote cell growth and proliferation. The ER-a receptor is known from studies on genetic knockout (KO) mice to be the major receptor involved in reproductive social behaviors, and in non-reproductive behaviors such as exploratory behavior, learning and memory, as well has having an important function in development.
This SGER project uses a novel approach to create an 'inducible' genetic KO mouse strain, rather than the simple knockouts currently available. The concept of an inducible knockout is not new, but there has been no reversible knockout developed for any of the steroid receptors, which have such widespread distribution and functional importance in the brain. The inducible genetic expression of ER-a will allow this steroid receptor function to be manipulated reversibly, and so turn the gene expression not only off, but also on again, during different stages of development and in adults, allowing exploration of whether there are distinct 'critical periods' in life for the function of this important receptor.
This is a high-impact/high-risk project because the outcome is not clear, but the potential impact of success is wide-ranging. If successful, the results will lead to further studies on time-dependent functions of steroid hormones, and will have a high impact extending beyond neuroendocrinology into the areas such as developmental biology and cognitive neuroscience. The project also provides a valuable training experience for a graduate student bridging molecular biology with neuroendocrinology.

[unreadable] DESCRIPTION (provided by applicant): Reproductive behaviors are typical of a variety of motivated behaviors that require integration of multiple internal and external cues for their successful execution. Gonadotropin releasing hormone (GnRH) is a master neuropeptide that coordinates reproductive condition and behavior. Yet, many animals, including humans, have a second form of GnRH, GnRH-II, which is highly conserved, present in brains of animals from all vertebrate classes (bony fish to humans), produced by a unique group of neurons and yet the peptide has no known function. In this new application we will test the novel hypothesis that this neuropeptide, GnRH-II, modulates reproductive behavior in response to the individual's nutritional status. Our hypothesis is based on our findings that; 1) Intracerebroventricular (icv) administration of GnRH-II, but not GnRH-I, peptide activates reproductive behavior in female musk shrews (Suncus murinus), but only in females experiencing caloric restriction; 2) Immunoreactivity of GnRH-II peptide varies with reproductive condition and nutritional status in female musk shrew brains, 3) Musk shrew brains contain specific type-II GnRH receptors which have a high affinity for GnRH-II peptide, and these receptors are found in nuclei known to control reproductive behavior, and 4) GnRH-II infusions suppress food consumption. In our laboratory we have the needed resources, experience with this animal model, and technical expertise to conduct these studies. We will use a combination of pharmacology, immunocytochemistry, surgical, and behavioral methods to build this new research program. The work proposed here will elucidate the function of GnRH-II in a novel and highly appropriate mammalian model system. The presence of the GnRH-II peptide in rodent brain is unclear, yet this peptide is widespread in human and non-human primates. By examining this neuropeptide first in a primitive mammal we will reveal the conserved and likely the most common function of this neuropeptide. This work employs several novel approaches to dissect the relationship between GnRH-II, feeding and sexual behaviors, and nutrition. It is likely that this information will provide new approaches for the study of GnRH-II peptide in humans. Moreover, this work may provide insights into physiological bases of eating disorders. [unreadable] [unreadable]

DESCRIPTION (provided by applicant): The Training Program in Neurobiology and Behavioral Development (NBDTP) at the University of Virginia is seeking funds for renewal and revitalization of the program. Funds are sought to train five PhD students working on dissertations relevant to neural and behavioral development, with the aim of producing the next generation of basic researchers able to both understand clinical needs and conduct translational work. To this end, the Program aims to have 19 Mentors from the College of Arts and Sciences and the School of Medicine, in five different departments, unified by participation in the interdisciplinary, degree-granting Neuroscience Graduate Program (NGP). The Training Program has been extensively revised with the addition of new and enthusiastic Faculty Mentors and a new, highly committed Director, all working towards implementing new training elements. The Program would now concentrate its energies by selecting only predoctoral students. These students would have already completed vigorous coursework in their first two years, attending weekly seminars, journal clubs and given opportunities to present their data. Recipients of the Training Program grant would be those who have shown an interest in translational work. One of the new goals is to have these trainees work with clinicians - by spending a semester doing clinical rotations and having a Clinical Mentor on their dissertation committee - new elements that will work to bridge the gap between basic research and applied medicine.

Across cultures, and species, males and females differ in the expression of aggression and parental nurturing behavior. Males are nearly universally the more aggressive, and the less parental, of the two sexes. The majority, but not all, sex differences in behavior can be attributed to differences in steroid hormone concentrations during development in males and females. Another potential source of differences between males and females are the genes that reside on the sex chromosomes. A transgenic mouse model in which gonadal and chromosomal sex are uncoupled is being employed to test this hypothesis. Two behavioral differences that can be attributed to sex chromosome complement, independent of gonadal sex, are offensive aggression and pup retreival behavior. One neural factor examined to date is correlated with these behaviors;vasopressin immunoreactivity in the lateral septum. In this new grant application the genetic underpinnings of these sex differences will be investigated. A series of genetic, behavioral, and pharmacological experiments will be conducted. First a unique mouse cross will be produced to determine if two copies of the X genes in the case of XX, or the presence of Y chromsome genes in XY females causes the differences between behaviors in XX and XY individuals. Next a set of studies will be done to examine the two behaviors of interest in more depth, and to quantify other sex differences in the brain in relation to sex chromosome complement. To determine if the exposure to androgens during development interacts with sex chromosome genes, androgens will be blocked in neonates just after birth. A genetic approach that takes advantage of a knock out mouse that cannot produce endogenous estrogens will be used as well. Neural vasopressin will be manipulated independently of sex chromosomes to determine how and if this neuropeptides is related to aggressive behavior. In sum this work will increase our understanding of the function of this important group of sexual dimorphic genes and their gene products. The work will also enrich and inform our knowledge of sex differences in both normal and clinical populations.

The award will support trainee-related activities at two annual meetings of the Society for Behavioral Neuroendocrinology (SBN). The annual conference brings together a diverse group of scientists studying the integration of genetic, cellular and molecular concepts into functional frameworks that improve our understanding of behavior. This grant supports significant initiatives to foster participation by undergraduate, graduate students, and post-doctoral fellows at society meetings. Funds will support the following: (1) Young Investigators Symposium. A selected group of graduate students and/or post-docs including substantial numbers of women present talks in a dedicated symposium. This format recognizes the accomplishments of trainees, facilitates their movement into more advanced positions, and motivates younger trainees. (2) Career Development Workshops. Workshops educate trainees on important career development topics and retain trainees in the sciences. (3) Travel Awards. Funding for travel to meetings is provided for the best trainees. This allows trainees to present their work in a supportive environment and gain exposure to the breadth and depth of the field. (4) Mentor-Mentee Lunches. Based on the professional and personal needs of the trainee, trainees will be matched with a senior Mentor. The pair will establish a long-term mentoring relationship. Trainees thus acquire a mentor outside of their advisor and local environment that will give them new insights into the research enterprise, how to balance life and work, and consult on career choices and options. (5) Trainee Poster Competition. Posters presented by undergraduates, graduate students, and post-doctoral fellows are judged, based on the scientific merit of the research, the effectiveness of the presentation and the trainee role in the research. The competition provides an opportunity to encourage and reward success at each level. Funding for trainee events at the SBN meeting will play a key role in the attraction and retention of the brightest and best-trained interdisciplinary scientists.

DESCRIPTION (provided by applicant): Project Summary The long term goal of this research program is to identify epigenetic mechanisms involved in sex differences in neurobehavioral diseases. Basic research on sex differences in behavior reveals two mechanisms. The best documented is differences in circulating levels of gonadal hormones in male versus female embryos and neonates which shape neuronal cell migration, connections and structures and are responsible for many adult behaviors. In addition, sex chromosome genes themselves are correlated with a number of sexually dimorphic behaviors. This latter mechanism has parallels in humans in which X-chromosome genes are linked to many mental disorders. Here we will ask whether the endocrine disrupting compound, bisphenol A (BPA), can modify behavior and if so whether it acts as a hypomethylator on candidate X-chromosome genes. In Aim 1 we will examine independent effects of sex chromosome and gonadal sex, in conjunction with BPA, on juvenile social behavior in mice. In Aim 2 we will conduct gene expression arrays to validate a set of candidate genes affected by BPA exposure during neural development. In the final aim we will ask if DNA methylation status in candidate gene promoters is affected by BPA and if histone methylation is likewise affected. We will use genetically engineered mice, molecular, genetic and behavioral methods to reveal epigenetic interactions between sex chromosome genes and BPA. The goal of our research is to find genes and processes that can help diagnose, treat and prevent mental illnesses. Understanding the epigenetic, as well as the genetic, bases for neurobehavioral diseases is essential for diagnosis, prevention and treatment. Here we focus on one environmental factor, bisphenol A, a man-made chemical that has the capacity to affect gene transcription through several mechanisms, and to which exposure during development may affect brain organization. Given the large sex differences in the prevalence of several neurobehavioral diseases (for example, autism is found 4 times more often in boys than in girls), we focus on epigenetic modification of mechanisms that underlie sex differences in behavior.

One of the most significant advances in Biology in the past 10 years is the development of the field of Epigenetics. Epigenetics literally means ?above the gene? and it describes a set of mechanisms whereby changes in gene structure can occur in an organism without any alternations to the DNA sequence, the building blocks of the genetic code. The mechanisms by which these changes occur are still unclear, but they include biochemical modifications to proteins that spool DNA into compacted spheres allowing the packaging of the long strands of DNA into cell nuclei. These changes are dynamic and are prevalent throughout embryonic development and can also occur in response to environmental stimuli. Another important process that occurs during embryonic development is the specification of gender differences in how male and female brains become organized. Early indications in this area of research suggest that factors that cause these brain changes are also part of the epigenetic machinery in cells, but few studies have brought these two research fields together in a cohesive manner. The immediate objective of this Research Coordinating Network (RCN) on Epigenetic Approaches to Sex Differences is to develop a structured framework whereby Behavioral and Molecular Biologists along with Bioinformaticians and Geneticists can collaborate directly using state-of-the art technology to address emerging questions focusing on the intersections between epigenetic mechanisms and gender differences of brain development. To achieve this we will hold a yearly meeting, develop a website to share information and protocols, and encourage students to move between laboratories to learn new techniques. The potential impact of the work includes novel insights into how the genders respond differentially to their environments. For example, women are apparently more susceptible to post-traumatic stress syndrome than men are after serving in combat. Thus, work from this RCN may help to explain how differences in cellular responses to stress influence important gender discrepancies in physiology and/or behavior.

DESCRIPTION (provided by applicant): Strong gender differences are well documented for many neural-motor diseases, yet, experimental work on motor systems rarely includes both genders and when it does, sex comparisons are not made. This gap is critical and will be addressed by the work in this proposal. The cerebellum is the master coordination center in the brain and its dysregulation produces many kinds of motor diseases. The ultimate goal of this research is to determine if mutations in sex chromosome genes play key roles in sexually dimorphic neurological motor disorders. The basis for the proposal is the observation that female mice, or mice of either sex with an XX sex chromosome complement, have more calbindin expression in the cerebellum than males or any XY individuals. Calbindin D28K is only present in the Purkinje cells, the only projection neurons in the cerebellum. This protein is linke with neuroprotection in the brain and reduced levels have been associated with many neurological diseases. The initial goal of this project is to determine if sex differences are the result of differences in numbers of Purkinje cells. The next question is to determine if there are sex differences dendritic structures in Purkinje cells. Finally we will determine if particular sub regions of the cerebellum are more sexually dimorphic than others. These goals will be accomplished with histological staining and morphometric analyses in addition to western blots and q RT-PCR. We will do all the studies in a mouse line allows separate analysis of sex chromosome complement and gonadal sex (i.e. ovaries and testes). Next, the genes that contribute to the sex differences will be revealed. This will be accomplished using a combination of engineered mouse models that enable cell-specific quantification of mRNA via gene expression microarray. Our hypothesis is that a sex chromosome gene(s) regulate calbindin and together these two factors modify Purkinje cell connections in a sexually dimorphic manner. This program offers a novel perspective on sex differences in cerebellar function based on sex chromosome genes; this will lead to innovations in diagnostics and interventions for patients with neurological motor diseases. PUBLIC HEALTH RELEVANCE: Neurological disorders strike an estimated 50 million Americans each year, and more of these patients are men than women. The research conducted in this program is specifically aimed at discovering the genetic bases for elevated vulnerability in men.

DESCRIPTION (provided by applicant): Many endocrine disrupting compounds (EDCs) have immediate effects on exposed individuals, but more complex concerns surround their long term actions on subsequent generations. The limited data from humans concur with the animal work suggesting that exposure to EDCs, particularly exposure during development, produce subtle increases in disease states, along with evidence for transgenerational effects. The precise epigenetic and/or genetic actions of EDCs on specific target genes, which produce stable modifications in subsequent generations, are unknown. The first goal of this program is to understand the mechanisms underlying transgenerational inheritance produced by human-relevant levels of the ubiquitous EDC, Bisphenol A (BPA). The work proposed is the first to test transgenerational actions of human-relevant exposures of BPA on brain and behaviors. Pregnant inbred mice are exposed to EDCs via voluntary ingestion of maternal diet. At birth, control and BPA pups are fostered to dams on control diet to isolate the actions of BPA to gestation and control for any consequences of these compounds on maternal behavior. We have shown that several social behaviors in the first and fourth generation (F4) of mice the BPA lineage differ significantly from control mice. Moreover, mRNA for two genes that regulate social behavior, vasopressin (Avp) and oxytocin (Oxt), are decreased in brains of the F4 BPA-exposed mice. Experiment one will determine the parent of origin for transmission of BPA's actions. In addition we will assess a number of cognitive and emotional behaviors in these mice to improve our ability to predict which human diseases may be sensitive to BPA. In the next study brains regions are probed with next generation sequencing techniques to establish target genes. Brains from F3 mice will be dissected into the nuclei that compose the social behavior network. A combination of Chromatin Immunoprecipitation (ChiP-) and RNA-sequencing will be conducted. These data provide targets for the epigenetic modifications found in brain. The mechanisms revealed will be applicable to other target tissues and thus a variety of diseases. This research plan will provide the field with a model for transgenerational actions of BPA and an epigenomic map of the consequences of these exposures.

Abstract This new program is a marriage of three important research fields; Drug Addiction, Sex Differences, and Environmental Health. Sex differences in drug-related behaviors have been well documented and attributed to actions of estrogens and progesterone in adult females. Here we move beyond this hypothesis and examine two other factors both of which have been implicated in reward via their actions on the dopamine system. Endocrine disruptors (EDCs) are ubiquitous in the environment and are known to alter sex differences in brain and behavior. EDC exposure is particularly potent when it occurs during neonatal development. As such it is a model for fetal origins of adult disease. This two-year grant will test the hypothesis that exposure to Bisphenol A (BPA), one of the well-studied and abundant EDCs in the environment, can increase vulnerability to cocaine. To further dissect the role of gender on drug vulnerability the four core genotype (FCG) mouse model will be used. The FCG allows separate analysis of gonadal hormones and sex chromosome complement (XX vs. XY). In aim one a dose-response study will be conducted with BPA exposure during gestation. In the next aim the FCG mice will be tested. Adult mice are fitted with chronic indwelling jugular cannula which allow them to self-administer cocaine. Fixed and progressive ratio schedules are used to examine self-administration and measure both acquisition and maintenance. The long term objectives of this work are to develop a research program to test the effects of environmental endocrine disrupting compounds in combination with gender, on motivated drug seeking and taking behavior.

Abstract Sex differences in drug-related behaviors have been well documented and attributed primarily to differences in levels of estrogens in adult men versus women. This program advances the field beyond this hypothesis to examine another important factor, sex chromosome complement, with a focus on X-chromosome genes that escape X-inactivation which are expressed in greater levels in female versus male brains. The four core genotype (FCG) mouse model allows separate analysis of gonadal hormones and sex chromosome complement (XX vs. XY). In Aim 1 these mice will be used to examine combined and separate actions of estradiol and sex chromosome complement on vulnerability to cocaine addiction using intravenous self- administration. Rates of acquisition and levels of motivation to obtain the drug will be evaluated. In Aim 2, we will define the role of two X-chromosome genes that escape inactivation in mouse and human brain, Utx and Smcx. These genes code for enzymes that demethylate histone modifications, regulating chromatin accessibility and transcription on a wide basis. This work will be conducted with two lines of inducible, tissue- specific knockout mice that lack the expression of either gene (Utx or Smcx) in CaMK2? in forebrain, including neurons in the nucleus accumbens. The mice will be tested as in Aim 1 for cocaine vulnerability and motivation. In Aim 3 brain tissue from the nucleus accumbens of mice tested in Aims 1 and 2 will be used for epigenetic analysis. A combination of ATAC (Assay for Transposase Accessible Chromatin-), Pro (Precision Nuclear Run-On-) and ChIP (Chromatin Immuno-Precipitation-) Sequencing will be used to identify transcriptionally active and repressed genes associated with chromatin restructuring and important transcription factors. The long term objective of this work is to reveal new mechanisms that underlie sex differences in vulnerability to addiction that help clinicians design sex-specific preventions/interventions.